The present invention relates to methods and reagents for typing HLA class I genes utilizing locus-specific nucleic acid amplification followed by sequence-specific detection of the amplified product.
The immune system has evolved a special mechanism to detect infections which occur within cells as opposed to infections which occur in extracellular fluid or blood, e.g., Janeway Jr., Scientific American, September, 73-79 (1993). Generally speaking, this mechanism acts in two steps: first, the immune system finds a way to signal to the body that certain cells have been infected, and then, it mobilizes cells specifically designed to recognize these infected cells and to eliminate the infection.
The initial step, signaling that a cell is infected, is accomplished by special molecules that deliver segments of the invading microbe to the outer surface of the infected cell. These molecules bind to peptide fragments of the invading microbe and then transport the peptides to the outside surface of the infected cell.
These transporter molecules are proteins of the major histocompatibility complex of genes, which in humans is referred to as the human leukocyte antigen complex, or HLA. These HLA molecules can be divided into two Classes: (i) class I molecules which are found on almost all types of cells, and (ii) class II molecules which appear only on cells involved in the immune response.
The two different classes of HLA molecules present peptides that arise in different places within cells. Class I molecules bind to peptides that originate from proteins in the cystolic compartment of the cell. After binding, the class I molecules fold around the foreign peptide then carry the peptide to the cell surface. The presentation of the peptide by the class I molecules then signals other cells of the immune system to destroy the host cell. The genes coding for the class I molecules are further subclassified as the HLA-A, HLA-B, and HLA-C genes.
Unlike class I molecules, class II molecules do not require peptide-directed folding to become active. Moreover, rather than signaling the destruction of the host cell, peptides presented by the class II molecules activate the internal defenses of the host cell, or alternatively, guide the synthesis of specific antibody molecules by the immune system.
The genes that encode the HLA molecules are among the most variable genes in humans: each variant coding for molecules which bind to different peptides. These genes are the same in all the cells of a particular individual, but differ from person to person.
HLA typing is performed routinely in connection with many medical indications, e.g., organ transplantation (rejection of organ grafts is believed to be greatly diminished if the HLA alleles of donor and recipient are identical), the study of auto-immune disease, and the determination of susceptibility to infectious disease.
Traditionally, the majority of HLA typing has been performed using serological techniques. However, these techniques have a number of serious drawbacks: (i) the availability of standard antisera is limited, (ii) the accuracy and resolution of the technique is limited by the small number of alleles which can be tested for, (iii) the speed of serological tests is very slow, and, (iv) new alleles can not be detected.
Recently, to solve many of the problems of serologically-based typing methods, molecular techniques have been employed for HLA typing, including restriction fragment length polymorphism analysis (RFLP), sequence specific oligonucleotide probing and/or priming techniques, and DNA sequencing. By looking directly at the genotype of the HLA system rather than the phenotype, the information content and accuracy of the typing procedure can be greatly enhanced. Examples of sequencing-based methods are provided by Santamaria et al., PCT/US92/01675; Holtz et al., DNA-Technology and Its Forensic Application, Berghaus et al. eds., p 79-84 (1991); Petersdorf et al., Tissue Antigens 44: 211-216 (1994); Guttridge et al., Tissue Antigens 44: 43-46 (1994); Santamaria et al., Human Immunology 37: 39-50 (1993); Santamaria et al., PCT/US92/01676; Santamaria et al., Human Immunology 33: 69-81 (1992); and Petersdorf et al., Tissue Antigens, 43: in press (1994). Examples of probing-based methods are provided by Bunce and Welsh, Tissue Antigens 43: 7-17 (1994); Anrien et al., Tissue Antigens 42: 480-487 (1993); Dominguez et al., Immunogenetics, 36: 277-282 (1992); Yoshida et al., Human Immunology 34: 257-266 (1992); Allen et al., Human Immunology 40: 25-32 (1994); Fernandez-Vina et al., Human Immunology 33: 163-173 (1992); and Teodorica and Erlich, EPO 92118396.8.
Important problems encountered in any of the above molecular techniques include the complexity, reliability and specificity of the DNA amplification procedures. These problems have become particularly critical as these molecular typing techniques have become more commonly used in the clinical environment. Because of the similarity among the HLA-A, -B, and -C genes, it has been up until very recently impossible to find amplification methods which allow discrimination between the three class I genes while at the same time are independent of (i) inadvertent amplification of closely related genes, e.g., neighboring pseudogenes, and (ii) independent of the extreme polymorphism found in these genes, e.g., Cereb et al., Tissue Antigens 45: 1-11 (1995). However, the protocol of Cereb relies on intronic primer sites, making it suseptable to promiscuous intronic mutations.
Current techniques have addressed these problems by limiting the generality of the methods, e.g., limiting the analysis to a subset of exons in a given gene, e.g., Petersdorf et al., Tissue Antigens, 44: 93-99 (1994), where the analysis requires multiple amplification primer sets and sequential amplifications to cover only the HLA-C subtype. This approach is not preferred because of the complexity of the protocols, the amount of information about the sample required prior to the analysis, and the number of reagents required.